1. Field of the Invention
The present invention relates to an enzyme, BgaC protein having beta-galactosidase (EC 3.2.1.23) activity derived from Streptococcus pneumoniae, and a method for using the same.
2. Description of the Related Art
Beta-galactosidase is a cleavage enzyme that belongs to family 35 of glycohydrolases, and that is found in plants and animals, as well as in a wide variety of microorganisms such as yeasts, fungi, bacteria, and archaea. Beta-galactosidase hydrolyzes lactose and its structurally related compounds, and additionally catalyzes transgalactosylation reactions of various beta-D-galactopyranosides including lactose. The hydrolase and transferase activities of beta-galactosidase have certain industrial applications (Nkayama and Amachi, 1999; Hung and Lee, 2002). Beta-galactosidase is widely used in the hydrolysis of lactose, which is present in milk products such as milk and whey, to glucose and galactose. The hydrolysis process is a fundamental method for reducing the lactose content employed in the food and dairy industries (Greenberg and Mahoney, Process Biochem. 16: 2-8, 1981; Gekas and Lopez-levia, 20: 2-12, 1985).
Meanwhile, many studies have been made on the relationship between the structure and function of a sugar chain in order to investigate its biological meaning and role, which has attracted a great deal of interest in the field. For these studies, there is a need for analysis of each sugar chain at the level of linkage specificity. In general, for the analysis of a component or sequence of a sugar chain, equipment such as HPLC and mass spectrometry has been widely used. However, a linkage-specific glycosidase is essentially needed for linkage-specifically analyzing the structure of a sugar chain. Therefore, there is a trial that employs a beta-galactosidase having linkage-specific glycosidase activity for analyzing the structure of a sugar chain.
Further, with respect to cancer treatment, there are a variety of treatment methods, such as chemotherapy administrating various anti-cancer agents, immunotherapy promoting antibody production against cancer cells, surgical therapy removing cancer cells, and radiation therapy killing cancer cells by irradiating radioactive rays. However, even though the primary cancer may be eradicated by such processes, some problems may still exist. That is, cancer may be a malignant tumor because of its metastatic ability, and in many cases, metastatic cancer is more likely to cause death. It cannot be said yet that a method for inhibiting the metastasis of cancer cells has been established, and a medicine having the effects of inhibiting the metastasis of cancer cells has not yet been commercially available. On the other hand, several steps are considered for the metastasis mechanism, and a casual relationship between the cancer metastasis and the sugar chain has been recently discussed in academic meetings. In the metastasis of cancer cells, cancer cells are first released from a cancer-developed site, and then move through the blood stream in a human body. E-selectin, which is one of the intercellular adhesive molecules, is expressed on the surface of an intravascular endothelial cell for several reasons. This E-selectin interacts with free cancer cells moving through the blood stream in the human body, and causes a rolling phenomenon, in which the free cancer cells roll on the surfaces of the intravascular endothelial cells and reduce their moving speed in the blood. Consequently, the free cancer cells adhere to the intravascular endothelial cells, and then pass through the intravascular endothelial cells. Thus, the cancer cells enter the vascular tissue, resulting in the formation of a new cancer cell nest.
In this series of steps, the adhesion between the E-selectin, which is one of the intercellular adhesive molecules expressed on the surface of the intravascular endothelial cell, and the sugar chains, which are present on the surfaces of cancer cells, plays a very important role in an initial stage of the adhesion between the cancer cells and the intravascular endothelial cells. As a sugar chain antigen on the surface of cancer cells, which interacts with the E-selectin, a sialic acid-containing complex sugar chain, called as Sialyl Lewis X (sLex) and Sialyl Lewis A (sLea), has been identified. That is, there is a report that the sugar chain acts as a ligand involved in the metastasis of cancer cells (Takada et al., Cancer Res. 53: 354-361, 1993). In the sugar chains, galactose-beta1,3-N-acetylglucosamine (Gal-β1,3-GlcNAc) is a core polysaccharide of sLea, and galactose-beta1,3-N-acetylglucosamine and galactose-beta1,3-N-acetylgalactosamine (Gal-β1,3-GalNAc) are major components of mucin-type and complex-type glycoproteins. Accordingly, a specific galactosidase capable of cleaving the terminal galactose at beta1,3 linkage is needed for inhibiting metastasis.
It has been known that BgaC protein has an activity of cleaving non-reducing terminal galactose linked by beta1,3 glycosidic linkage. However, BgaC proteins, which are galactosidases capable of cleaving the galactose linked by beta1,3-linkage, have not been well known, until now. Among the proteins, there is no enzyme that specifically recognizes a sugar followed by a galactose to cleave the galactose.
On the other hand, when an infectious microorganism invades a host cell, its glycosidase is involved in the cleavage of sugar chains exposed on the surface of the host cell. Therefore, the related genes are often found in pathogenic microorganisms. There is a report that a galactosidase having the activity of cleaving beta 1,4 linkage, which is designated as BgaA, was found in Streptococcus pneumoniae causing pneumonia. The Streptococcus pneumoniae BgaA is a putative 2,235-amino acid protein having a molecular weight of 247.3 kDa, which is present on the cell surface, and purified from culture medium (Zahner and Hakenberck, J. Bacteriol. 182: 5919-5921, 2000; Glasgow et al., J. Biol. Chem. 252: 8615-8623, 1977; Hughes and Jeanloz, Biochemistry, 10: 1535-1548, 1964).
The present inventors have analyzed the genomic information of Streptococcus pneumoniae that has already been disclosed (Hoskins et al., J. Bacteriol. 183: 5709-5712, 2001). As a result, it was found that another new putative nucleic acid molecule having galactosidase activity exists, thereby performing functional analysis of the nucleic acid molecule. Consequently, a novel cleavage enzyme, which has different sugar chain specificity from BgaA, was detected.
In particular, it was found that the novel enzyme selectively recognizes only the sugar chain having galactose-beta1,3-N-acetylglucosamine linkage, and hydrolyzes the galactose, which is useful for the functional analysis and modification of a sugar chain.
Further, it was observed that treatment of the enzyme inhibits the colony formation of cancer cells, which offers a possibility of using the enzyme as an anti-cancer agent, thereby completing the present invention.